The present invention generally relates to three-dimensional (3-D) structural data of protein molecules, and to methods for their determination.
Proteins differ from one another in their sequence of amino acids, which typically results in folding of the protein molecule into a unique 3-D structure referred to as its tertiary structure. The overall shape of this 3-D structure controls the basic function of the protein, and therefore the discovery of its tertiary structure, or the quaternary structure of its complexes, can provide important clues about how the protein functions. X-ray crystallography is one of the major methods used to determine protein structures, but requires that the protein be capable of crystallization in preparation for diffraction analysis. Solved structures are usually deposited in the protein data bank (PDB).
Due to the growing number of new protein targets for crystallization and entry of their protein crystal structures into the PDB, the need for high-throughput technologies for protein crystal analysis has emerged. Currently, numerous imaging modalities are employed for scoring protein crystal “hits” during the crystallization condition screening process. Several instances of fluorescence imaging techniques such as two-photon excited UV fluorescence (TPE-UVF), UV laser-stimulated fluorescence (UVF), covalent modification of the protein with a fluorophore, and noncovalent fluorescent molecule intercalation have appeared in the literature. TPE-UVF utilizes intrinsic fluorescent properties of aromatic residues, e.g., tryptophan, found in most proteins to provide contrast. However, this technique is limited to proteins with high intrinsic fluorescence. For proteins that do not exhibit strong intrinsic fluorescence (due to a lack of tryptophan), such as insulin, UVF has been used for imaging. However, exposing protein crystals to a UV-C band excitation laser has been shown to damage disulfide bonds within the protein. By covalently modifying proteins with fluorophores, researchers have been able to circumvent the need for intrinsic fluorescence and damaging laser wavelengths to visualize proteins. However, this method adds a further sample handling step, and studies have shown that covalent modification can adversely influence the folding structure of the protein. All of the above mentioned techniques suffer from a major limitation due to a lack of selectivity for crystalline protein, producing nonzero signals from crystalline proteins, amorphous protein aggregates, and proteins in solution.
Second harmonic generation (SHG) microscopy has been shown to be a complementary technique for selective imaging of protein crystals. SHG, or the frequency doubling of light, can arise when noncentrosymmetric crystalline material is illuminated by sufficiently intense light, with negligible contributions from disordered media such as protein aggregate. Recently, SHG microscopy has been successfully developed for automated analysis of 96-well plates, assessing crystal quality through polarization analysis, and rapid centering of crystals on a synchrotron beamline. However, current SHG microscopy techniques can only detect an estimated 84% of known protein crystal structures with crystals exhibiting SHG activities spanning several orders of magnitude. For smaller crystals or for high-throughput screening applications with shorter signal integration times, this coverage value will be correspondingly reduced, suggesting the need for improvements in the signal to noise ratio (SNR, or S/N) for crystal detection. The SHG response varies considerably depending upon the protein, the protein orientation within the lattice, and the symmetry of the lattice. The latter of which is the main driving force behind the inability to detect roughly 16% of the current PDB entries. A detailed discussion of the relationships between symmetry, structure, and SHG activity of proteins can be found in Haupert et al., Acta Crystallographica Section D 68, p. 1513-1521 (2012), whose contents are incorporated herein by reference.
In view of the above, improving the detection limits of SHG would be desirable to increase the coverage of protein crystals amenable to SHG detection, reduce the time frame required for protein crystal imaging by SHG, and allow detection of increasingly smaller crystals at early stages of crystallization.